The assembly/disassembly and enzymatic activities of protein nanomachines underlie all cellular functions, and dysregulated nanomachines are the ultimate culprits in cancer. Our proposed research seeks to establish a new conceptual framework to specifically understand the cellular organization of molecular activities. We hypothesize that cellular biochemical activities are spatially organized into an activity architecture via the specific organization of active molecules and their regulatory partners. This activity architecture, together with the structural and mechanical architecture of the cell, encodes all the information needed to drive cellular function. We further hypothesize that perturbations to this activity architecture, even by a few dysregulated driver molecules, could lead to detrimental effects on cellular functions such as loss of control over cell growth, divisio and death. The key technological innovation of our research program is to develop and utilize a new generation of enabling technologies for visualizing and perturbing biochemical and biophysical processes in the native environment of a living cell. Enabled by these new technologies, we will start testing our general hypothesis by elucidating the spatial organization of the enzymatic activities of protein kinases, a family of enzymes that play critical roles in normal cell physiology and tumorigenesis. We will further probe how this kinase activity architecture is dynamically modulated by hormones and growth factors and perturbed by oncogenic mutations. We will also use our single- molecule optogenetic method to probe the connectivity, robustness and sensitivity of the activity architecture. We expect the successful completion of this study will yield a suite of new, transformative technologies with the potential to change the way that biochemical processes are studied in the context of cellular organization. Most importantly, establishing this new conceptual framework of a spatially organized activity architecture should produce a paradigm shift with respect to our understanding of the behaviors of active molecules in their native environment. Characterization of dys-organized activity architectures in cancers should lead to the development of more effective therapeutic treatments that target such dysregulation.